Using two-dimensional gel electrophoresis (2DE), an atlas was constructed showing the location and relative concentration of a number of different proteins from 25 distinct neuroanatomical regions of the male rat brain. Using this as a basis, a number of different projects were initiated. First, we have shown that the concentration of two distinct proteins is different in the medial preoptic area of male and female rats. Second, we have shown that disruption of the cholinergic innervation to the hippocampus and the occipital cortex affects the concentration of 4 different proteins. This suggests that the amounts of these polypeptides in brain are regulated by the cholinergic nervous system. Third, we have initiated a series of studies using human cortical tissue and have shown the following: A) The apparent concentration of a number of different proteins changes shortly after death, suggesting that results obtained using post-mortem human brain tissue should be interpreted with caution. B) Irradiation as a treatment for various carcinomas of the CNS apparently alters the concentration of a small number of proteins in the surrounding normal tissue. C) Different types of CNS tumors have protein patterns which are both distinct and different from that seen in normal cortical tissues. Fourth, we have succeeded in identifying a number of heretofore unidentified proteins on two-dimension gels of rat and human cortex. These include the isomers of the glycolytic enzyme enolase, glial fibrillary acidic protein, alpha and beta tubulin and a number of neurofilament proteins. Finally, we have shown that proteins from discrete nuclei in fresh brain tissue can be labeled with [35S]-methionine in vitro. These proteins can then be separated by 2DE and radiolabel uptake estimated by autofluorography. These results provide an estimate of amino acid incorportation into protein during short-term tissue culture experiments.